Nonsense-mediated mRNA decay (NMD) in mammalian cells provides a mechanism whereby mRNAs that prematurely terminate translation, i.e., encode potentially deleterious truncated proteins, are degraded. Therefore, NMD can be viewed as an important means of quality control. For reasons that remain unclear, the majority of mammalian mRNAs that prematurely terminate translation is subject to NMD at a point when they co-purify with nuclei, while a minority is subject to NMD in the cytoplasm. During the past funding period, our studies of mRNA encoding the selenoprotein glutathione peroxidase (GPx) 1 have lent important insight into the mechanism of cytoplasmic NMD. As examples, we have found that cytoplasmic NMD is restricted to newly synthesized mRNA and takes place during what we call a "pioneer" round of translation. This proposal aims to take these exciting studies further. In Aim1, the polarity and enzymology of NMD will be determined using (i) silencing RNAs to down-regulate the levels of proteins involved in general mRNA decay, (ii) immunoprecipitation to assay for interactions between NMD factors and proteins involved in general mRNA decay, and (iii) RT-PCR and RNase mapping to measure the relative rates at which mRNA 5' and 3' ends are degraded during NMD. Aim 2 will characterize the pioneer translation initiation complex by (i) identifying component proteins and their interactions, (ii) analyzing translational efficiency relative to that of the steady-state initiation complex, (iii) assessing mRNPs harboring RNA localization signals (zip codes) after proper localization as evidence for or against the possibility that these mRNAs are first translated only after proper localization, and (iv) determining if NMD and/or the decay of mRNA in general are coordinated with decay of the encoded protein. Aim 3 will examine the translational dependence and kinetics of remodeling the pioneer initiation complex to the steady-state initiation complex. Aim 4 will identify what determines the cellular site of NMD by analyzing hybrid or mutated mRNAs and mRNP proteins. Insight into NMD and the pioneer translation initiation complex will provide information on basic cellular metabolism that can be used in the design of therapies for the many diseases that are due to the lack of full-length protein because of the premature termination of translation and NMD.